Composition and device for in vivo cartilagerepair

ABSTRACT

The composition as described serves for in vivo cartilage repair. It basically consists of a naturally derived osteoinductive and/or chondroinductive mixture of factors (e.g. derived from bone) or of a synthetic mimic of such a mixture combined with a nanosphere delivery system. A preferred mixture of factors is the combination of factors isolated from bone, known as BP and described by Poser and Benedict (WO 95/13767). The nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter (whereby the majority of particles preferably ranges between 200-400 nm) in which nanospheres the combination of factors is encapsulated. The nano-spheres are loaded with the mixture of factors in a weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have a release profile with an initial burst of 10 to 20% of the total load over the first 24 hours and a long time release of at least 0.1 per day during at least seven following days. The nanospheres are composed of e.g. ((D,L)-lactic acid/glycolic acid)-copolymer (PLGA). The loaded nanospheres are e.g. made by phase inversion. The composition is advantageously utilized as a device comprising any biodegradable matrix in which the nanospheres loaded with the factor combination is contained.

BACKGROUND OF THE INVENTION

[0001] Articular cartilage, an avascular tissue found at the ends ofarticulating bones, has no natural capacity to heal. During normalcartilage ontogeny, mesenchymal stem cells condense to form areas ofhigh density and proceed through a series of developmental stages thatends in the mature chondrocyte. The final hyaline cartilage tissuecontains only chondrocytes that are surrounded by a matrix composed oftype II collagen, sulfated proteoglycans, and additional proteins. Thematrix is heterogenous in structure and consists of threemorphologically distinct zones: superficial, intermediate, and deep.Zones differ among collagen and proteoglycan distribution,calcification, orientation of collagen fibrils, and the positioning andalignment of chondrocytes (Archer et al., J. Anat. 189(1): 23-35, 1996;Morrison et al., J. Anat. 189(1): 9-22 1996, Mow et al., Biomaterials13(2): 67-97, 1992). These properties provide the unique mechanical andphysical parameters to hyaline cartilage tissue.

[0002] In 1965, a demineralized extraction from bovine long bones wasfound to induce endochondral bone formation in the rat subcutaneousassay (Urist Science 150: 893-899, 1965). Seven individual factors,termed Bone Morphogenetic Proteins (BMPs), were isolated to homogeneityand, because of significant sequence homology, classified as members ofthe TGFβ super-family of proteins (Wozney, et al., Science 242: 1528-34,1988; Wang et al., Proc. Nat. Acad. Sci. 87: 2220-2224, 1990). Theseindividual, recombinantly-produced factors also induce ectopic boneformation in the rat model (Luyten et al., J. Biol. Chem. 264: 13377-80,1989; Celeste et al., Proc. Nat. Acad. Sci. 87: 9843-50, 1990). Inaddition, in vitro tests have demonstrated that both BMP-2 and TGFβ-1induce mesenchymal stem cells to form cartilage (Denker, et al.,Differentiation 59(1): 25-34, 1995; Denker et al., 41st Ann. Orthop.Res. Society 465: 1995). Both BMP-7 and BMP-2 have been shown to enhancematrix production of chondrocytes in vitro (Flechtenmacher J. ArthritisRheum. 39(11): 1896-904, 1996: Sailor et al., J. Orthop. Res. 14:937-945, 1996). From these data we can conclude that not only are theBMPs important regulators of osteogenesis, but that they also playcrucial roles during chondrogenic development in vitro.

[0003] A partially-purified protein mixture from bovine long bones,termed BP (Bone Protein), also induces cartilage and bone formation inthe rat subcutaneous assay (Poser and Benedict, WO95/13767). BP incombination with calcium carbonate promotes bone formation in the body.In vitro, BP induces mesenchymal stem cells to differentiatespecifically to the cartilage lineage, in high yields, and to latestages of maturation (Atkinson et al., J. Cellular Biochem. 65: 325-339,1997).

[0004] The molecular mechanism for cartilage and bone formation has beenpartially elucidated. Both BMP and TGFβ molecules bind to cell surfacereceptors (the BMP/TGFβ receptors), which initiates a cascade of signalsto the nucleus that promotes proliferation, differentiation tocartilage, and/or differentiation to bone (Massague Cell 85: 947-950,1996).

[0005] In 1984, Urist described a substantially pure, but notrecombinant BMP, combined with a biodegradable polylactic acid polymerdelivery system for bone repair (U.S. Pat. No. 4,563,489). This systemblends together equal quantities of BMP and polylactic acid (PLA) powder(100 μg of each) and decreases the amount of BMP required to promotebone repair.

[0006] Hunziker (U.S. Pat. No. 5,368,858; U.S. Pat. No. 5,206,023)describes a cartilage repair composition consisting of a biodegradablematrix, a proliferation and/or chemotactic agent, and a transformingfactor. A two stage approach is used where each component has a specificfunction over time. First, a specific concentration ofproliferation/chemotactic agent fills the defect with repair cells.Secondly, a larger transforming factor concentration transforms repaircells into chondrocytes. Thereby the proliferation agent and thetransforming agent may both be TGFβ differing in concentration only. Inaddition, the patent discloses a liposome encapsulation method fordelivering TFGβ-1 serving as transformation agent.

[0007] Hattersley et al. (WO 96/39170) disclose a two factor compositionfor inducing cartilaginous tissue formation using a cartilageformation-inducing protein and a cartilage maintenance inducing protein.Specific recombinant cartilage formation inducing protein(s) arespecified as BMP-13, MP-52, and BMP-12, and cartilagemaintenance-inducing protein(s) are specified as BMP-9. In oneembodiment, BMP-9 is encapsulated in a resorbable polymer system anddelivered to coincide with the presence of cartilage formation inducingprotein(s).

[0008] Laurencin et al., (U.S. Pat. No. 5,629,009) disclose achondrogenesis-inducing device, consisting of a polyanhydride andpolyorthoester, that delivers water soluble proteins derived fromdemineralized bone matrix, TGFβ, EGF, FGF, or PDGF.

[0009] The results of the approaches to cartilage repair as cited aboveare encouraging but they are not satisfactory. In particular, the repairtissue arrived at is not fully hyaline in appearance and/or it does notcontain the proper chondrocyte organization. Furthermore, previousapproaches to cartilage repair have been addressed to very small defectsand have not been able to solve problems associated with repair oflarge, clinically relevant defects.

[0010] One reason that previous approaches failed to adequately repaircartilage may be that they were not able to recapitulate naturalcartilage ontogeny faithfully enough, this natural ontogeny being basedon a very complicated system of different factors, factor combinationsand factor concentrations with temporal and local gradients. A singlerecombinant growth factor or two recombinant growth factors may lack theinductive complexity to mimic cartilage development to a sufficientdegree and/or the delivery systems used may not have been able to mimicthe gradient complexity of the natural system to a satisfactory degree.

[0011] Previous approaches may also have failed because growth factorconcentrations were not able to be maintained over a sufficient amountof time, which would prevent a full and permanent differentiation ofprecursor cells to chondrocytes. The loss of growth factor could becaused by diffusion, degradation, or by cellular internalization thatbypasses the BMP/TGFβ receptors. Maintaining a sufficient growth factorconcentration becomes particularly important in repair of large sizeddefects that may take several days or several weeks to fully repopulatewith cells.

[0012] The object of this invention is to create a composition forimproved cartilage repair in vivo. The inventive composition is toenable in vivo formation of repair cartilage tissue which tissueresembles endogenous cartilage (in the case of articular cartilage withits specific chondrocyte spatial organization and superficial,intermediate, and deep cartilage zones) more closely than repair tissueachieved using known compositions for inducing cartilage repair. Afurther object of the invention is to create a device forn cartilagerepair which device contains the inventive composition.

[0013] This object is achieved by the composition and the device asdefined by the claims.

BRIEF DESCRIPTION OF THE INVENTION

[0014] The inventive composition basically consists of a naturallyderived osteoinductive and/or chondroinductive mixture of factors (e.g.derived from bone) or of a synthetic mimic of such a mixture combinedwith a nanosphere delivery system. A preferred mixture of factors is thecombination of factors isolated from bone, known as BP and described byPoser and Benedict (WO 95/13767). The nanosphere delivery systemconsists of nanospheres defined as polymer particles of less than 1000nm in diameter (whereby the majority of particles preferably rangesbetween 200-400 nm) in which nanospheres the combination of factors isencapsulated. The nanospheres are loaded with the mixture of factors ina weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) andhave an analytically defined release profile (see description regardingFIG. 2) showing an initial burst of 10 to 20% of the total load over thefirst 24 hours and a long time release of at least 0.1 per day during atleast seven following days, preferably of 0.1 to 1% over the following40 to 60 days. The nanospheres are composed of e.g. (lacticacid-glycolic acid)-copolymers (Poly-(D,L)lactic acid-glycolic acid)made of 20 to 80% lactic acid and 80 to 20% of glycolic acid, morepreferably of 50% lactic acid and 50% of glycolic acid.

[0015] The loaded nanospheres are e.g. made by phase inversion accordingto Mathiowitz et al. (Nature, 386: 410-413, 1997) or by other methodsknown to those skilled in the art (Landry, Ph.D Thesis, Frankfurt,Germany).

[0016] The inventive composition is advantageously utilized as a devicecomprising any biodegradable matrix including collagen type I and II,and hyaluronic acid in which matrix the nanospheres loaded with thefactor combination is contained. The matrix can be in the form of asponge, membrane, film or gel. The matrix should be easily digestible bymigrating cells, should be of a porous nature to enhance cell migration,and/or should be able to completely fill the defect area without anygaps.

[0017] It is surprisingly found that the inventive compositionconsisting of an osteoinductive and/or chondroinductive combination offactors (e.g. derived from natural tissue) encapsulated in nanospheresas specified above, if applied to a defect area of an articularcartilage, leads to the transformation of virtually all precursor cellsrecruited to the repair area to chondrocytes, and furthermore, leads toa homogeneous chondrocyte population of the repair area and to achondrocyte order and anisotropic appearance as observed in endogenoushyaline cartilage. These findings encourage the prospect that theinventive composition may lead to significant improvements alsoregarding repair of large defects.

[0018] As mentioned above, instead of an osteoinductive and/orchondroinductive mixture of factors derived from bone (BP), theinventive composition may comprise natural factor mixtures derived fromother tissues (e.g. cartilage, tendon, meniscus or ligament) or may evenbe a synthetic mimic of such a mixture having an osteoinductive and/orchondroinductive effect. Effective mixtures isolated from natural tissueseem to contain a combination of proliferation, differentiation, andspatial organizing proteins which in combination enhance the tissuerebuilding capacity more effectively than single proteins (e.g.recombinant proteins).

[0019] The specified, analytically defined release profile of suchfactor mixtures from nanospheres results in the formation ofconcentration gradients of proliferation and differentiation factors,which obviously mimics the complex gradients of factors observed duringnatural development very well. The nanosphere extended release profileis sufficient to provide growth factor during the time frame that repaircells arrive into the matrix. The release profile obviously leads to ahomogeneous population of a matrix with precursor cells, to fulldifferentiation of virtually all of the precursor cells to chondrocytes,and to the formation of an endogenous hyaline cartilage structure.

[0020] Another advantage of the inventive composition is that when thenanospheres are placed in a matrix to form a device for cartilagerepair, they are randomly distributed and remain in place when in ajoint cartilage defect. During cellular infiltration anddifferentiation, the nanospheres are in the correct position over thecorrect time frame.

[0021] Nanospheres have been demonstrated to adhere to thegastrointestinal mucus and cellular linings after oral ingestion(Mathiowitz et al., Nature, 386 410-413 1997). We envisage thatnanospheres also adhere to cartilage precursor cells and furthermore,may also adhere to BMP/TGFβ receptors located on the cell membrane. Thisproperty allows localized high-efficiency delivery to the target cellsand/or receptors. Because of the nanosphere small size and the chemicalproperties, they are more effective than liposomes or diffusioncontrolled delivery systems. The efficient delivery to the receptorswill facilitate chondrogenesis.

[0022] Derived from the above findings, we envisage the followingmechanism for cartilage repair using the effect of the inventivecomposition. During the first 24 hours (initial burst) 10 to 20% of thetotal load of the factor mixture is released from the nanospheres intothe matrix and diffuses into the synovial environment. Following theinitial burst, the nanospheres begin to release factors at a slow rate,which produces gradients of proliferation, differentiation, and spatialorganizing proteins. In response to such gradients, precursor cellsmigrate to the defect site. The loaded nanospheres adhere to cartilageprecursor cells and to the BMP and TGFβ receptors to provide localizedhighly efficient delivery. The precursor cells become differentiated tochondrocytes and secrete type II collagen and cartilage-specificproteoglycans. The composition of the present invention stimulatesdifferentiation of virtually all of these cells to overt chondrocytesand induces an ordered cartilage structure which closely resembleshyaline cartilage. Furthermore, we envisage that this release systemwill allow homogeneous repair of large defect sites and repair ofdefects from patients with low quantities of precursor cells.

[0023] For in vivo cartilage repair, the inventive device consisting ofa matrix and the loaded nanospheres is placed in a chondral lesion thatwas caused by trauma, arthritis, congenital, or other origin. The damagecan result in holes or crevices or can consist of soft, dying, or sickcartilage tissue that is removed surgically prior to implantation of thedevice. Because of the unique properties of the inventive deviceprecursor cells populate the matrix, differentiate to chondrocytes, andform hyaline cartilage.

[0024] Application of the inventive composition (without matrix) e.g. byinjection can be envisaged also, in particular in the case of smalldefects. Thereby at least 2 μg of the composition per ml of defect sizeis applied or at least 20 ng of the osteoinductive and/orchondroinductive mixture encapsulated in the nanospheres is applied perml defect size.

[0025] The inventive composition and the inventive device are suitablefor repair of cartilage tissue in general, in particular for articularcartilage and for meniscus cartilage.

BRIEF DESCRIPTION OF THE FIGURES

[0026] The following figures illustrate the physical and chemicalparameters of the inventive composition, the in vitro cartilageinductive activity of BP released from nanospheres and in vivo repair ofan articular cartilage defect using the inventive device.

[0027]FIG. 1 shows a scanning electron micrograph of BP-loadednanospheres;

[0028]FIG. 2 shows the release profile (cumulative release vs. time) ofthe inventive composition;

[0029]FIG. 3 shows the release profile of the inventive compositioncompared with release profiles of nanosphere delivery systems loadedwith other proteins;

[0030]FIG. 4 shows the volume of a cartilage defect vs. the daysrequired for populating the defect with repair cells;

[0031]FIG. 5 shows micromass cultures in the presence or absence ofnanospheres loaded with BP;

[0032]FIG. 6 shows cartilage marker analyses for in vitro culturescontaining BP only and for similar cultures containing the inventivecomposition;

DETAILED DESCRIPTION OF THE INVENTION

[0033]FIG. 1 shows a scanning electron micrograph of BP-loadednanospheres. The microparticle sizes range from 100-1000 nm with themajority of individual particles ranging between 200-400 nm.

[0034] The release rate profile of the inventive composition wasdetermined by in vitro analysis of BP delivered from nanospheres. Thesenanospheres were made by phase inversion according to the method asdisclosed by Mathiowitz et al. (Nature 386, 410-414, 1997) of((DL)lactic acid/glycolic acid)-copolymer containing the two acids in aweight ratio of 50:50 and they were loaded with 1% and with 4% of BP.

[0035] For determination of the release rate profile, the nanosphereswere placed in a sterile saline solution and incubated at 37° C. BPreleased into the supernatant was measured using a BCA assay (Pierce).BP released from the nanospheres as specified shows two successive anddistinct profile parts: a fast release (initial burst) of approximately10 to 20% of the loaded BP over the first 24 hours and a slow release of0.1 to 1% per day (cumulative 40% to 50%) over 40 to 60 days (FIG. 2).

[0036] The release is intermediate between zero-order and first-orderkinetics. Both the 1% and 4% encapsulated BP have similar releaseprofiles.

[0037] For attaining release rate profiles as specified above and asnecessary for the improved results in cartilage repair the nanospheresare to be adapted accordingly when using factor mixtures other than BP.Thereby, e.g the composition of the nanosphere copolymer, the molecularweight of the polymer molecules and/or the loading percentage of thenanospheres may be changed. The optimum nanosphere character for eachspecific case has to be found experimentally whereby the release rateprofile is analyzed in vitro as described above.

[0038] In the same way, the nanosphere delivery system can be modifiedregarding the percentage of BP to be released in the first 24 hours,percentage of BP to be released after 24 hours and/or length of timeafter the first 24 hours during which the remainder of BP is released.In addition, the percentage of BP loaded to the nanospheres is of coursevariable too, whereby for obtaining the results as described for thespecified composition, all the modifications are to be chosen such thatthe resulting delivery keeps within the range as specified.

[0039] All of the above parameters can be modified to account for thepatient's age, sex, diet, defect location, amount of blood present inthe defect, and other clinical factors to provide optimal cartilagerepair. For example, nanospheres with longer release rates are used fortreating larger defects and/or for patients with fewer precursor cells(e.g. older patients or patients with degenerative symptoms). Incontrast, patients with larger quantities of progenitor cells and/orsmaller defects may require a shorter release rate profile.

[0040]FIG. 3 shows the release profile as shown in FIG. 2 fornanospheres as specified above loaded with BP and with other proteins(same loading percentages) such as BSA (bovine serum albumin) orlysozyme. The drastically different release characteristics shows thatthe profile is dependent on the protein type also. The same is valid fora more hydrophobic mixture of bovine bone derived proteins (PIBP).

[0041]FIG. 3 illustrates the singularity of the inventive combinationconsisting of the specific delivery system (nanospheres as specifiedabove encapsulating the factors) and the specific protein mixture (BP)which is obviously the key to the improved results in cartilage repairas observed when using the inventive composition or device.

[0042] To determine the length of time required for precursor cellrepopulation of different sized defects, the following calculation wasperformed. We estimate that approximately 50,000 cells are recruited tothe defect/day. Since the cellular density of cartilage is about 4×10⁷cells/ml, a 10 μl volume defect will take approximately 8 days to fillwith cells. FIG. 4 plots the number of days required to fill differentvolume defects with cells. The Figure assumes an infinite supply ofcells and a constant rate of cell attraction to the defect site. Thegraph demonstrates that the larger a defect size is, the more time isrequired to completely fill it with cells. Since a 60 μl volume defectwill take over 45 days to fill, this Figure demonstrates the necessityfor a long term release of factors to induce differentiation of theprecursor cells over up to a two month period.

[0043] To determine whether BP bioactivity is harmed by theencapsulation process and to determine whether the released BP was fullybioactive, the following assay was performed. Previously, it wasdemonstrated that 10T1/2 micromass cultures exposed to BP induceformation of a three dimensional spheroid structure that can be observedmacroscopically in tissue culture wells (Atkinson et al., J. CellularBiochem. 65: 325-339, 1997). BP concentrations equal or greater than 20ng/ml were required for spheroid formation. No spheroid forms in theabsence of BP or at concentrations less than 10 ng/ml (see followingtable). In this assay, 10T1/2 mesenchymal stem cells act as in vitromodels for the precursor cells recruited to a natural defect.

[0044] We employed the same assay to test the bioactivity of BP releasedfrom 1% loaded nanospheres. BP was eluted from nanospheres at 37° C. ina 5% CO₂ humidified incubator. After 24 hours 16% BP is released; andbetween 24 hours and 7 days, 7% BP was released (FIG. 2). Thesupernatant was collected, serial dilutions were made, and thesupernatant was added to 10T1/2 micromass cultures. BP released fromnanospheres at both time points formed spheroids at concentrationsgreater than 20 ng/ml, but not at concentrations between 0 and 10 ng/ml(see following table). Non-encapsulated BP also formed spheroids atconcentrations greater than 20 ng/ml, but not at concentrations between0 and 10 ng/ml. We conclude that both nanosphere encapsulation and,release of BP does not inhibit BP bioactivity.

[0045] Spheroid formation (+=no spheroid formation;+=spheroidformation): BP concentration (ng/ml) state of used BP 0-10 20-1000non-encapsulated BP − + released from nanospheres (24 h) − + releasedfrom nanospheres (168 h) − +

[0046] To determine the effect of BP slow release in the direct presenceof micromass cultures, the following assay was performed. Nanosphereswere washed for 24 hours and the supernatant was discarded. Thenanospheres were then added to micromass cultures at a quantity suchthat 10 or 25 ng/ml of BP would be released over 24 hours. Release of 25ng/ml resulted in spheroid formation whereas release of 10 ng/ml did notform spheroids (FIG. 5). Similarly, the addition of 10 ng ofnon-encapsulated BP per ml did not form a spheroid whereas the additionof 25 ng of non-encapsulated BP per ml did form a spheroid. Regardingthe specific in vitro set-up, we conclude that slow release of BP over24 hours is as effective as a single dose of BP.

[0047] To determine whether the BP released from nanospheres was aschondrogenic as non-encapsulated BP, spheroids were analyzed for type IIcollagen and proteoglycan content. 10T1/2 spheroids from the above assaythat had formed with 1 μg of released BP per ml or 1 μg ofnon-encapsulated BP per ml were tested histologically with Azure and H+Estains and immunocytochemically with antibodies to type II collagenafter 7 days. Both encapsulated and non-encapsulated BP inducedcartilage markers such as type II collagen, proteoglycan, and round cellshape (FIG. 6). In addition, no qualitative differences were observedbetween encapsulated and non-encapsulated BP with respect to cellquantity, viability, morphology, or organization (FIG. 6). We concludethat BP retains full chondrogenic capacity after release fromnanospheres.

[0048] The in vitro models used for determining the chondroinductiveeffect of BP differ from the in vivo case by the fact that in the invitro case the precursor cells are present in an appropriate number andin an appropriate distribution whereas in the in vivo case the precursorcells first have to populate the defect and for this reason have tomigrate into the defect. Only in the latter case and for achievingrepair cartilage which resembles natural cartilage to a high degree, itis essential for the BP to be released over a prolonged time periodaccording to a specific release profile.

EXAMPLE

[0049] The following example shows that BP released from nanospheresinduces cartilage repair in chondral defects in vivo whereby virtuallyall cells recruited to the defect become chondrocytes, whereby the cellstructure obtained is ordered, and whereby a hyaline matrix is built up.

[0050] Using a sheep model, unilateral defects of 0.5 mm width, 0.5 mmdepth and 8 to 10 mm length were created in the trochlear groove of thepatella. The defects did not penetrate the subchondral bone. The sheepemployed in this study were seven years old and displayed degenerativesymptoms, including brittle bones, chondromalacia, and subchondralcysts. Because of their advanced age and degenerative symptoms, theseamimals probably have decreased numbers of precursor cells. The defectswere then dressed according to Hunziker and Rosenberg (J. Bone JointSurg. 78A(5): 721-733, 1996) with minor changes. Briefly, afterenzymatic proteoglycan removal with Chondroitinase AC, 2.5 μl of asolution containing 200 units Thrombin per ml was placed in the defect.Then, a paste was filled into the defect, the paste containing per ml:60 mg Sheep Fibrinogen (Sigma), 88 mg Gelfoam (Upjohn) and either 10 μgof BP-nanospheres or 10 μg of BP-nanospheres plus 80 ng rhIGF-1 (R+DSystems).

[0051] The nanospheres used were the nanospheres as specified in thedescription regarding FIG. 2 and they were loaded with 1% (w/w) of BP.

[0052] Assuming that the in vitro determined release rate isapproximately the same as for the in vivo case, 10 to 20 ng BP per mlwere released during the first 24 hours and approximately 0.1 to 1 ngper day for the following approximately 60 days.

[0053] After eight weeks, necropsies were performed. The repairedcartilage histology showed that virtually all of the precursor cellswere differentiated to chondrocytes throughout the defect. In addition,there was an ordered cartilage appearance with cells on the top beingmore flattened morphologically than cells in the center and with thepresence of ordered, stacked chondrocytes in the lowest zone. Therepaired cartilage was fully integrated into the endogenous tissue. Inaddition, the cartilage repaired with only BP-nanospheres was notsignificantly different from the cartilage repaired using BP-nanospheresplus IGF-1.

[0054] In conclusion, these results demonstrate that BP released fromnanospheres is sufficient for cartilage repair and that no addintionalfactor is required (such as e.g recombinant factor IGF-1). Using theinventive device constitutes a one step method for cartilage repair,whereby the nanosphere release of BP is sufficient for differentiationof virtually all of the precursor cells to chondrocytes and forinduction of an ordered cartilage structure.

[0055] Other Publications:

[0056] Archer C W, Morrison E H, Bayliss M T, Ferguson M W: Thedevelopment of articular cartilage: II. The spatial and temporalpatterns of glycosaminoglycans and small leucine-rich proteoglycans; JAnat (ENGLAND) 189 (Pt 1): 23-35 (1996)

[0057] Atkinson B L, Fantle, K S, Benedict J J, Huffer W E,Gutierrez-Hartmann A: A Combination of Osteoinductive Bone ProteinsDifferentiates Mesenchymal C3H/10T1/2 Cells Specifically to theCartilage Lineage; J. Cellular Biochem. 65: 325-339 (1997).

[0058] Celeste A J, Iannazzi J A, Taylor R C, Hewick R M, Rosen V, WangE A, Wozney J M: Identification of transforming growth factor betafamily members present in bone-inductive protein purified from bovinebone; Proc Natl Acad Sci USA, December, 87(24): 9843-7 (1990)

[0059] Denker A E, Nicoll S B, Tuan R S: Formation of cartilage-likespheroids by micromass cultures of murine C3H10T1/2 cells upon treatmentwith transforming growth factor β1′; Differentiation 59(1): 25-34 (1995)

[0060] Denker A E, Nicoll S B, Tuan R S: 41st Annual Meeting Orthop.Res. Society. (abstract): 465 (1995)

[0061] Flechtenmacher J, Huch K, Thonar E J, Mollenhauer J A, Davies SR, Schmid T M, Puhl W, Sampath T K, Aydelotte M B, Kuettner K E:Recombinant human osteogenic protein 1 is a potent stimulator of thesynthesis of cartilage proteoglycans and collagens by human articularchondrocytes; Arthritis Rheum, November, 39(11): 1896-904 (1996)

[0062] Hunziker E B and Rosenberg L C: Repair of Partial-ThicknessDefects in Articular Cartilage: Cell Recruitment from the SynovialMembrane; J. Bone Joint Surgery 78-A(5): 721-733 (1996)

[0063] Kim S, Turker M S, Chi E Y, Sela S, Martin G M: Preparation ofmultivesicular liposomes; Bioch. et Biophys. Acta 728:339-348 (1983)

[0064] Landry F B: Degradation of Poly (D,L-lactic acid) Nanoparticlesin artificial gastric and intestinal fluids; in vivo uptake of thenanoparticles and their degradation products; Thesis for the Dept. ofBiochemistry, Pharmacy, and Food Chemistry of the Johann Wolfgang GoetheUniversity in Frankfurt, Germany

[0065] Luyten F P, Cunningham N S, Ma S, Muthukumaran N, Hammonds R G,Nevins W B, Woods W I, Reddi A H: Purification and partial amino acidsequence of osteogenin, a protein initiating bone differentiation; JBiol Chem, 264(23): 13377-80 (1989)

[0066] Massague J: TGFβ Signaling: Receptors, Transducer, and MadProteins; Cell 85: 947-950 (1996)

[0067] Mathiowitz E, Jacob J S, Jong Y S, Carino G P, Chickering D E,Chaturvedi P, Santos C A, Vijayaraghavan K, Montgomery S, Bassett M,Morrell C: Biologically erodable microspheres as potential oral drugdelivery systems; Nature 386: 410-4 (1997)

[0068] Morrison E H, Ferguson M W, Bayliss M T, Archer C W: Thedevelopment of articular cartilage: I. The spatial and temporal patternsof collagen types; J Anat (ENGLAND) 189(Pt 1): 9-22 (1996)

[0069] Mow V C, Ratcliff A, Poole A R: Cartilage and diarthrodial jointsas paradigms for hierarchical materials and stuctures; Biomaterials13(2): 67-97 (1992)

[0070] Sailor L Z, Hewick R M, Morris E A: Recombinant human bonemorphogenetic Protein-2 maintains the articular chondrocyte phenotype inlong-term culture; J. Orthop. Res. 14: 937-945 (1996)

[0071] Urist M R: Bone: formation by autoinduction; Science 150: 893-899(1965)

[0072] Wang E A, Rosen V, D'Alessandro J S, Bauduy M, Cordes P, HaradaT, Israel D I, Hewick R M, Kerns K M, LaPan P, Luxenberg D P, McQuaid D,Moutsatsos I, Nove J, Wozney J M: Recombinant human bone morphogeneticprotein induces bone formation;' Proc Natl Acad Sci USA, 87(6): 2220-4(1990)

[0073] Wozney J M, Rosen V, Celeste A J, Mitsock L M, Whitters M J, KrizR W, Hewick R M, Wang E A: Novel Regulators of bone formation: molecularclones and activities; Science 242: 1528-34 (1988)

1. Composition for inducing in vivo cartilage repair comprising anosteoinductive and/or chondroinductive mixture of factors derived fromnatural tissue or a sythetic mimic of such a mixture encapsulated innanospheres, whereby the nanospheres are polymer particles having a sizeof less than 1000 nm and an in vitro analytically determined releaserate profile with an initial burst of 10 to 20% of the total load overthe first 24 hours and a long time release of at least 0.1% per dayduring at least seven following days and whereby the nanospheres areloaded with between 0.001 and 17% weight percent of the mixture offactors.
 2. Composition according to claim 1, characterized in that thelong term release is between 0.1 and 1% of the total load per day overbetween 40 and 70 days.
 3. Composition according to claim 1 or 2,characterized in that the osteoinductive and/or chondroinductive mixtureof factors is derived from bone, cartilage, tendon, meniscus orligament.
 4. Composition according to claim 1 or 2, characterized inthat the osteoinductive and/or chondroinductive mixture of factors isthe mixture known as BP (bone protein) derived from bovine long bonesand partly purified.
 5. Composition according to claim 4, characterized,in that the nanospheres are loaded with between 1 and 4% weight percentof BP.
 6. Composition according to one of claims 1 to 5, characterizedin that the nanospheres consist of ((D,L)lactic acid/glycolicacid)-copolymer containing 20 to 80% of lactic acid and 80 to 20% ofglycolic acid.
 7. Composition according to one of claims 1 to 5,characterized in that the ((D,L)lactic acid/glycolic acid)-copolymercontains 50% of lactic acid and 50% of glycolic acid.
 8. Compositionaccording to one of claims 1 to 7, characterized in that the nanospheresare made by phase inversion.
 9. Device containing the compositionaccording to one of claims 1 to 8 and further comprising a porousbiodegradable matrix.
 10. Device according to claim 9, characterized inthat it contains at least 2 μg of loaded nanospheres per ml.
 11. Deviceaccording to claim 9, characterized in that it contains at least 20 ngof the osteoinductive and/or chondroinductive mixture of factors per ml.12. Device according to one of claims 9 to 11, characterized in that thematrix has the form of a sponge, membrane, film or gel.
 13. Deviceaccording to one of claims 9 to 12, characterized in that the matrixconsists of collagen type I, collagen type II or hyaluronic acid. 14.Use of the composition according to one of claims 1 to 8 for preparing adevice for in vivo cartilage repair.
 15. Use according to claim 14,characterized in that the cartilage is articular cartilage or meniscuscartilage.
 16. Use of the composition according to one of claims 1 to 8for cartilage repair on an animal with a degenerative disease.
 17. Useof the device according to one of claims 9 to 13 for cartilage repair onan animal with a degenerative disease.
 18. Method for in vivo cartilagerepair comprising the step of filling a cartilage defect with a deviceaccording to one of claims 9 to
 13. 19. Method according to claim 18,characterized in that the defect is dressed before filling.
 20. Methodfor in vitro cartilage repair comprising the step of applying to thecartilage defect a composition according to one of claims 1 to
 8. 21.Method according to claim 20, characterized in that the composition isapplied by injection.
 22. Method according to claim 20 or 21,characterized in that the composition is applied in an amount of atleast 2 μg per ml defect size.
 23. Method according to claim 20 or 21,characterized in that the composition is applied in an amount such thatthe osteoinductive and/or chondroinductive mixture is present in thedefect in an amount of at least 20 ng per ml defect size.